DESCRIPTION(Adapted from applicant's abstract): The overall objective of this application is to elucidate the biochemical functions of E.C.3.4.24.15 [EP24.15], the prototypical member of the mammalian family of endopeptidases that process/modulate neuropeptides. EP24.15 is present in brain, pituitary, and gonads, and has been directly implicated in critical physiologic pathways and neuroendocrine/neurodegenerative disorders and cancer. This enzyme processes neuropeptides extracellularly and thus is crucial in regulating signal transduction events essential to cell growth and differentiation. Understanding the function of EP24. 15 and its interaction with substrates and inhibitors will provide unique insight into aspects of normal and disease states and will aid in the design of new therapeutic agents for a variety of disorders. Experiments in this application will integrate biochemical, genetic, theoretical, structural and cell biological approaches to studying the enzyme-ligand interactions of this metalloenzyme. These strategies are also applicable to other macromolecules. Therefore, the interrelated specific aims of this project are: Specific Aim 1. Determinatlon of elements responsible for substrate binding and catalvsis in EP24.15 EP24. 15 protein will be functionally characterized utilizing structural genetics and kinetic determinations. Aim la. What amino acid deterrninants are responsible for the catalysis of neuropeptides? Aim lb. Which residues comprise the substrate [and inhibitor] binding subsites of the enzyme? Specific Aim 2. Define components modulatingr EP24. 15 activitv. localization. and substrate size selectivity. 4.15 is phosphorylated in vitro/in vivo, present in the nucleus, and though an endooligopeptidase, can cleave some large substrates. Aim 2a. Determine the effect of phosphorylation upon EP24. 15 enzyme activity. Aim 2b. What are the structural determinants of the enzyme which translocate it to the nucleus? Aim 2c. EP24. 15 is defined as an 'endooligopeptidase'. What is the mechanism of size selectivity? Specific Aim3. Modeling and higher resolution studies of EP24. 15 Structural biological approaches: molecular modeling of homologous domains and diffraction analyses. Aim 3a. Build a prototype from bacterial homologues for this class of mammalian enzymes . Aim 3b. Execute multiwavelength anomalous diffraction analyses of EP24. 15.